Sterol Esterase

We analyzed the relative basal mRNA expression levels of lipogenic and lipolytic genes in epiploic visceral adipose tissue (VAT) and abdominal subcutaneous adipose tissue (SAT). Five grams of VAT and SAT were obtained during bariatric surgery in the morbidly obese patients [1] (link), [32] (link). The biopsy samples were washed in physiological saline and immediately frozen in liquid nitrogen. Biopsy samples were maintained at −80°C until analysis. Frozen adipose tissue was homogenized with an Ultra-Turrax 8 (Ika, Staufen, Germany). Total RNA was extracted using RNeasy lipid tissue midi kit (QIAGEN Science, Hilden, Germany), and total RNA was treated with 55URNasefree deoxyribonuclease (QIAGEN) following the manufacturer's instructions. The purity of the RNA was determined by the absorbance260/absorbance280 ratio on the Nanodrop. The integrity of total purified RNA was checked by denaturing agarose gel electrophoresis and ethidium bromide staining. Total RNA was reverse transcribed to cDNA by using a high-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems, Foster City, CA). The cDNA was used for quantitative real-time PCR with duplicates. We analyzed the relative basal mRNA expression levels of peroxisome proliferator-activated receptor-ã (PPARã) (Hs00234592_m1, RefSeq. NM_005037.5, NM_015869.4, NM_138711.3 and NM_138712.3), acyl Coenzyme A:cholesterol acyltransferase (DGAT1) (Hs00201385_m1, RefSeq. NM_012079.4), aquaporin 7 (AQP7) (Hs00357359_m1, RefSeq. NM_001170.1), acetyl-CoA carboxylase 1 (ACC1) (Hs00167385_m1, RefSeq. NM_198834.1, NM_198836.1, NM_198837.1, NM_198838.1 and NM_198839.1), acetyl-CoA synthetase short-chain family member 2 (ACSS2) (Hs00218766_m1, RefSeq. NM_001076552.2, NM_018677.3 and NM_028046.1), ATP citrate lyase (ACL) (Hs00982738_m1, RefSeq. NM_001096.2 and NM_198830.1), phosphoenolpyruvate carboxykinase (PEPCK) (Hs00159918_m1, RefSeq. NM_002591.3), glycerol kinase (GyK) (Hs02340011_g1, RefSeq. NM_000167.4 and NM_203391.2), adipose triglyceride lipase (ATGL) (Hs00386101_m1, RefSeq. NM_020376.3), hormone-sensitive lipase (HSL) (Hs00193510_m1, RefSeq. NM_005357.2), and perilipin (Hs00160173_m1, RefSeq. NM_001145311.1 and NM_002666.4). We choose these genes because, previously, we have study the association between these lipogenic and lipolitic genes with basal anthropometric and biochemical variables [1] (link). The cycle threshold (Ct) value for each sample was normalized with the expression of PPIA (Hs99999904_m1). There is no variation in the expression of this housekeeping gene in the condition tested. Real-time quantitative PCR was performed on a 7900HT Fast real-time PCR system using commercial Taqman assays (Applied Biosystems) [33] (link). SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems) were used to analyze the results with the comparative Ct method (2−ΔΔCt). All data were expressed as an n-fold difference relative to the calibrator (a mixture of the SAT and VAT tissues was used as calibrator sample).

The total RNA in each liver sample was extracted using Trizol following the manufacturer’s instructions (Accurate Biology, Changsha, China). The concentrations and purities of total RNA were determined by Spectrophotometer (Denovix DX-11, Wilmington, DE, USA). Then, the cDNA was obtained by reverse transcription kit (Accurate Biology, Changsha, China). The mRNA relative expressions of fatty-acid synthase (FASN), peroxisome proliferator activated receptor gamma (PPAR-γ), solid alcohol regulatory element binding protein 1c (SREBP1c), CCAAT/enhancer binding proteins alpha (C/EBPα), adipose triglyceride lipase (ATGL), lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and sirtuin1 (Sirt1) in the liver tissue were determined by using a LightCycler 96 fast real-time PCR system (Roch, Switzerland) and SYBR^®Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Dalian, China), as previously described by Li et al. [20 (link)]. The primer sequences are presented in Table S1. The target gene’s relative mRNA expression was calculated using the threshold cycle ( $2^{-\Delta \Delta \mathrm{Ct}}$ ) method and normalized by the intrinsic reference gene (β-actin) expression.

For bile acid analysis [30 (link)], 1 μL of bile sample was diluted with 99 μL of Milli-Q^® water and then incubated with the reagent solution (6 mg NAD, 0.5 M hydrazine hydrate buffer, 0.05 M Na-pyrophosphate) for 4 min. The mix was then incubated with the solution (0.03 M Tris-EDTA; 0.3 U/mL 3-alpha-OH steroid dehydrogenase), and the bile acid concentration was determined by spectrophotometry (excitation of 340/330 nm/emission of 440/420 nm, CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany). For phospholipid (PL) analysis, 1 μL of bile sample was diluted with 49 μL of Milli-Q^® water and then incubated with the reagent solution (100 mM MOPS, pH 8; 0.55 mM HVA; 20 mM CaCl₂; 11 U/mL phospholipase-D; 1.66 U/mL peroxidase; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (1 M MOPS, pH 8, 50 U/mL choline oxidase), and the PL concentration was determined by spectrophotometry (excitation 340/330 nm/emission 440/420 nm using a CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany). For cholesterol analysis, 1 μL of bile sample was diluted with 29 μL of Milli-Q^® water and then incubated with the reagent solution (100 mM MOPS, pH 8, 0.25 mM HVA; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (0.1 M MOPS, pH 8, 0.06 U/mL cholesterol oxidase, 0.15 U/mL cholesterol esterase, 0.45 U/mL peroxidase, 0.06 mM taurocholate), and the cholesterol concentration was determined by spectrophotometry (excitation of 330/340 nm/emission of 420/440 nm, CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany).